Liquid, aqueous pharmaceutical compositions of factor VII polypeptides

ABSTRACT

The present invention is directed to liquid, aqueous pharmaceutical compositions stabilized against chemical and/or physical degradation containing Factor VII polypeptides, and methods for preparing and using such compositions, as well as vials containing such compositions, and the use of such compositions in the treatment of a Factor VII-responsive syndrome. The main embodiment is represented by a liquid, aqueous pharmaceutical composition comprising at least 0.01 mg/mL of a Factor VII polypeptide (i); a buffering agent (ii) suitable for keeping pH in the range of from about 4.0 to about 9.0; and at least one stabilizing agent (iii) comprising a —C(═N—Z 1 —R 1 )—NH—Z 2 —R 2  motif, e.g. benzamidine compounds and guanidine compounds such as arginine.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation U.S. application Ser. No.12/617,471, now issued as U.S. Pat. No. 8,026,214, filed Nov. 12, 2009which is a continuation of U.S. application Ser. No. 11/353,335, nowissued as U.S. Pat. No. 7,732 405, tiled Feb. 14, 2006, which is acontinuation (filed under 35 USC §120) of International PatentApplication PCT/DK2004/000537 (published as WO 2005/016365), filed Aug.12, 2004, which designated the US, and further claims the benefit ofpriority to U.S. Provisional Patent Application 60/496,443, filed Aug.20, 2003; Danish Patent Application PA 2003 01161, filed Aug. 14, 2003;and International Patent Application PCT/DK04/000181, filed Mar. 18,2004 under 35 USC §119, the entirety of each of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to liquid, aqueous pharmaceuticalcompositions containing Factor VII polypeptides, and methods forpreparing and using such compositions, as well as containers containingsuch compositions, and the use of such compositions in the treatment ofa Factor VII-responsive syndrome. More particularly, the inventionrelates to liquid compositions stabilized against chemical and/orphysical degradation.

BACKGROUND OF THE INVENTION

A variety of Factors involved in the blood clotting process have beenidentified, including Factor VII (FVII), a plasma glycoprotein.Coagulation is initiated by the formation of a complex between TissueFactor (TF) being exposed to the circulating blood following an injuryto the vessel wall, and FVIIa which is present in the circulation in anamount corresponding to about 1% of the total FVII protein mass. FVIIexists in plasma mainly as a single-chain zymogen which is cleaved byFXa into its two-chain, activated form, FVIIa. Recombinant activatedFactor VIIa (rFVIIa) has been developed as a pro-haemostatic agent. Theadministration of rFVIIa offers a rapid and highly effectivepro-haemostatic response in haemophilic subjects with bleedings, whocannot be treated with other coagulation Factor products due to antibodyformation. Also bleeding in subjects with Factor VII deficiency orsubjects having a normal coagulation system but experiencing excessivebleeding can be treated successfully with FVIIa.

It is desirable to have administration forms of Factor VIIa suitable forboth storage and for delivery. Ideally, the drug product is stored andadministered as a liquid. Alternatively, the drug product islyophilized, i.e. freeze-dried, and then reconstituted by adding asuitable diluent prior to patient use. Ideally, the drug product hassufficient stability to be kept in long-term storage, i.e. more than sixmonths.

The decision to either maintain the finished drug product as a liquid orto freeze-dry it is usually based on the stability of the protein drugin those forms. Protein stability can be affected inter alia by suchfactors as ionic strength, pH, temperature, repeated cycles offreeze/thaw, and exposures to shear forces. Active protein may be lostas a result of physical instabilities, including denaturation andaggregation (both soluble and insoluble aggregate formation), as well aschemical instabilities, including, for example, hydrolysis, deamidation,and oxidation, to name just a few. For a general review of the stabilityof protein pharmaceuticals, see, for example, Manning, et al.,Pharmaceutical Research 6:903-918 (1989).

While the possible occurrence of protein instabilities is widelyappreciated, it is impossible to predict particular instability problemsof a particular protein. Any of these instabilities can result in theformation of a protein by-product, or derivative, having loweredactivity, increased toxicity, and/or increased immunogenicity. Indeed,protein precipitation may lead to thrombosis, non-homogeneity of dosageform and amount, as well as clogged syringes. Furthermore,post-translational modifications such as, for example, gammacarboxylation of certain glutamic acid residues in the N-terminus andaddition of carbohydrate side chains provide potential sites that may besusceptible to modification upon storage. Also, specific to Factor VIIa,being a serine protease, fragmentation due to autocatalysis may occur(enzymatic degradation). Thus, the safety and efficacy of anycomposition of a protein is directly related to its stability.Maintaining stability in a liquid form is generally different frommaintaining stability in a lyophilized form because of highly increasedpotential for molecular motion and thereby increased probability ofmolecular interactions. Maintaining stability in a concentrated form isalso different from the above, because of the propensity for aggregateformation at increased protein concentrations.

When developing a liquid composition, many factors are taken intoconsideration. Short-term, i.e. less than six months, liquid stabilitygenerally depends on avoiding gross structural changes, such asdenaturation and aggregation. These processes are described in theliterature for a number of proteins, and many examples of stabilizingagents exist. It is well-known that an agent effective in stabilizingone protein actually acts to destabilize another. Once the protein hasbeen stabilized against gross structural changes, developing a liquidcomposition for long-term stability (e.g., greater than six months)depends on further stabilizing the protein from types of degradationspecific to that protein. More specific types of degradation mayinclude, for example, disulfide bond scrambling, oxidation of certainresidues, deamidation, cyclization. Although it is not always possibleto pinpoint the individual degradation species, assays are developed tomonitor subtle changes so as to monitor the ability of specificexcipients to uniquely stabilize the protein of interest.

It is desirable that the pH of the composition is in a physiologicallysuitable range upon injection/infusion, otherwise pain and discomfortfor the patient may result.

For a general review of protein compositions, see, for example, Clelandet al.: The development of stable protein compositions: A closer look atprotein aggregation, deamidation and oxidation, Critical Reviews inTherapeutic Drug Carrier Systems 1993, 10(4): 307-377; and Wang et al.,Parenteral compositions of proteins and peptides: Stability andstabilizers, Journal of Parenteral Science and Technology 1988(Supplement), 42 (2S).

Factor VIIa undergoes several degradative pathways, especiallyaggregation (dimerisation), oxidation, and autolytic cleavage (clippingof the peptide backbone or “heavy chain degradation”). Furthermore,precipitation may occur. Many of these reactions can be slowedsignificantly by removal of water from the protein. However, thedevelopment of an aqueous composition for Factor VIIa has the advantagesof eliminating reconstitution errors, thereby increasing dosingaccuracy, as well as simplifying the use of the product clinically,thereby increasing patient compliance. Ideally, compositions of FactorVIIa should be stable for more than 6 months over a wide range ofprotein concentrations. This allows for flexibility in methods ofadministration. Generally, more highly concentrated forms allow for theadministration of lower volumes, which is highly desirable from thepatients' point of view. Liquid compositions can have many advantagesover freeze-dried products with regard to ease of administration anduse.

Today, the only commercially available, recombinantly-made FVIIpolypeptide composition is a freeze-dried Factor FVIIa product which isreconstituted before use; it contains a relatively low Factor VIIaconcentration, e.g., about 0.6 mg/mL. A vial (1.2 mg) of NovoSeven®(Novo Nordisk A/S, Denmark) contains 1.2 mg recombinant human FactorVIIa, 5.84 mg NaCl, 2.94 mg CaCl₂, 2 H₂O, 2.64 mg glycylglycine(GlyGly), 0.14 mg polysorbate 80, and 60.0 mg mannitol; it isreconstituted to pH 5.5 by 2.0 mL water for injection (WFI). Whenreconstituted, the protein solution is stable for use for 24 hours.Thus, no liquid ready-for-use- or concentrated Factor VII products arecurrently commercially available.

Accordingly, it is an objective of this invention to provide a liquid,aqueous Factor VII polypeptide pharmaceutical composition which providesacceptable control of chemical and/or physical degradation products suchas enzymatic degradation or autocatalysis products.

SUMMARY OF THE INVENTION

The present inventors have discovered that Factor VII or analoguesthereof (“Factor VII polypeptides”), when formulated as liquid, aqueouspharmaceutical compositions together with at least one stabilizing agent(iii) comprising a —C(═N—Z¹—R¹)—NH—Z²—R² motif exhibit improvedstability and thereby allow for prolonged storage before actual use.

Thus, one aspect of the present invention relates to a liquid, aqueouspharmaceutical composition comprising

at least 0.01 mg/mL of a Factor VII polypeptide (i);

a buffering agent (ii) suitable for keeping pH in the range of fromabout 4.0 to about 9.0; and

at least one stabilizing agent (iii) comprising a —C(═N—Z¹—R¹)—NH—Z²—R²motif, wherein

Z¹ and Z² independently are selected from the group consisting of —O—,—S—, —NR^(H)— and a single bond, where R^(H) is selected from the groupconsisting of hydrogen, C₁₋₄-alkyl, aryl and arylmethyl, and R¹ and R²independently are selected from the group consisting of hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, optionally substituted heterocyclyl, or

Z² and R² are as defined above and —C═N—Z¹—R¹ forms part of aheterocyclic ring, or

Z¹ and R¹ are as defined above and —C—NH—Z²—R² forms part of aheterocyclic ring, or

—C(═N—Z¹—R¹)—NH—Z²—R² forms a hetercyclic ring wherein —Z¹—R¹—R²—Z²— isa biradical.

A second aspect of the present invention relates to a method forpreparing a liquid, aqueous pharmaceutical composition of a Factor VIIpolypeptide, comprising the step of providing the Factor VII polypeptide(i) at a concentration of at least 0.01 mg/mL in a solution comprising abuffering agent (ii) suitable for keeping pH in the range of from about4.0 to about 9.0; and at least one stabilizing agent (iii) comprising a—C(═N—Z¹—R¹)—NH—Z²—R² motif, wherein

Z¹ and Z² independently are selected from the group consisting of —O—,—S—, —NR^(H)— and a single bond, where R^(H) is selected from the groupconsisting of hydrogen, C₁₋₄-alkyl, aryl and arylmethyl, and R¹ and R²independently are selected from the group consisting of hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, optionally substituted heterocyclyl, or

Z² and R² are as defined above and —C═N—Z¹—R¹ forms part of aheterocyclic ring, or

Z¹ and R¹ are as defined above and —C—NH—Z²—R² forms part of aheterocyclic ring, or

—C(═N—Z¹—R¹)—NH—Z²—R² forms a hetercyclic ring wherein —Z¹—R¹—R²—Z²— isa biradical.

A third aspect of the present invention relates to the liquid, aqueouspharmaceutical composition for use as a medicament.

A fourth aspect of the present invention relates to the use of theliquid, aqueous pharmaceutical composition for the preparation of amedicament for treating a Factor VII-responsive syndrome.

A fifth aspect of the present invention relates to a method for treatinga Factor VII-responsive syndrome, the method comprising administering toa subject in need thereof an effective amount of the liquid, aqueouspharmaceutical composition.

A sixth aspect of the present invention relates to an air-tightcontainer containing the liquid, aqueous pharmaceutical composition andoptionally an inert gas.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention resides in the development ofa novel stabilized liquid, aqueous pharmaceutical composition comprisinga Factor VII polypeptide. More specifically, the liquid, aqueouspharmaceutical composition comprises

at least 0.01 mg/mL of a Factor VII polypeptide (i);

a buffering agent (ii) suitable for keeping pH in the range of fromabout 4.0 to about 9.0; and

at least one stabilizing agent (iii) comprising a —C(═N—Z¹—R¹)—NH—Z²—R²motif, wherein

Z¹ and Z² independently are selected from the group consisting of —O—,—S—, —NR^(H)— and a single bond, where R^(H) is selected from the groupconsisting of hydrogen, C₁₋₄-alkyl, aryl and arylmethyl, and R¹ and R²independently are selected from the group consisting of hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, optionally substituted heterocyclyl, or

Z² and R² are as defined above and —C═N—Z¹—R¹ forms part of aheterocyclic ring, or

Z¹ and R¹ are as defined above and —C—NH—Z²—R² forms part of aheterocyclic ring, or

—C(═N—Z¹—R¹)—NH—Z²—R² forms a hetercyclic ring wherein Z¹ R¹ R² Z² is abiradical.

The term “C₁₋₆-alkyl” is intended to encompass acyclic and cyclicsaturated hydrocarbon residues which have 1-6 carbon atoms and which canbe linear or branched. Particular examples are methyl, ethyl, n-propyl,isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,cyclopropylmethyl, n-pentyl, isopentyl, n-hexyl, etc. Similarly, theterm “C₁₋₄-alkyl” encompasses acyclic and cyclic saturated hydrocarbonresidues which have 1-4 carbon atoms and which can be linear orbranched.

Similarly, the term “C₂₋₆-alkenyl” is intended to encompass acyclic andcyclic hydrocarbon residues which have 2-6 carbon atoms and comprise oneunsaturated bond, which can be linear or branched. Examples ofC₂₋₆-alkenyl groups are vinyl, allyl, but-1-en-1-yl, but-2-en-1-yl,pent-1-en-1-yl, and hex-1-en-1-yl.

The term “optionally substituted” in connection with C₁₋₆-alkyl andC₂₋₆-alkenyl groups is intended to denote that the group in question maybe substituted one or several times, preferably 1-3 times, with group(s)selected from the group consisting of hydroxy, C₁₋₆-alkoxy (i.e.C₁₋₆-alkyl-oxy), C₂₋₆-alkenyloxy, oxo (forming a keto or aldehydefunctionality), aryl, aryloxy, arylcarbonyl, heterocyclyl,heterocyclyloxy, heterocyclylcarbonyl, amino, mono- anddi(C₁₋₆-alkyl)amino, halogen, where any aryl and heterocyclyl may besubstituted as specifically described below for optionally substitutedaryl and heterocyclyl.

“Halogen” includes fluoro, chloro, bromo, and iodo.

When used herein, the term “aryl” is intended to denote a fully orpartially aromatic carbocyclic ring or ring system, such as phenyl,naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl,benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferredexample.

The term “heterocyclyl” is intended to denote a saturated, partiallyunsaturated, partially aromatic or fully aromatic carbocyclic ring orring system where one or more of the carbon atoms have been replacedwith heteroatoms, e.g. nitrogen (═N— or —NH), sulphur (—S—), and/oroxygen (—O—) atoms. Examples of such heterocyclyl groups are oxazolyl,oxazolinyl, oxazolidinyl, isoxazolyl, isoxazolinyl, isoxazolidinyl,oxadiazolyl, oxadiazolinyl, oxadiazolidinyl, thiazolyl, iso-thiazolyl,pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl,piperidinyl, coumaryl, furyl, quinolyl, benzothiazolyl, benzotriazolyl,benzodiazolyl, benzoxozolyl, diazolyl, diazolinyl, diazolidinyl,triazolyl, triazolinyl, triazolidinyl, tetrazol, etc. Preferredheterocyclyl groups are 5-, 6- or 7-membered monocyclic groups such asisoxazolyl, isoxazolinyl, oxadiazolyl, oxadiazolinyl, pyrrolyl,pyrrolinyl, diazolyl, diazolinyl, triazolyl, triazolinyl, imidazolyl,imidazolinyl, etc.

The term “heterocyclic ring” is intended to mean a ring corresponding tothose defined under “heterocyclyl”.

In connection with the terms “aryl”, “heterocyclyl” and “heterocyclicring”, the term “optionally substituted” is intended to denote that thegroup in question may be substituted one or several times, preferably1-3 times, with group(s) selected from hydroxy (which when present in anenol system may be represented in the tautomeric keto form), C₁₋₆-alkyl,C₂₋₆-alkenyl, phenyl, benzyl, C₁₋₆-alkoxy, oxo (which may be representedin the tautomeric enol form), carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino,dihalogen-C₁₋₄-alkyl, trihalogen-C₁₋₄-alkyl, and halogen. The mosttypical examples of substituents are hydroxyl, C₁₋₄-alkyl, phenyl,benzyl, C₁₋₄-alkoxy, oxo, amino, mono- and dimethylamino and halogen.

Besides the fact that R¹ and R² independently can be selected from thegroup consisting of hydrogen, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substituted aryl,optionally substituted heterocyclyl, it is also possible that a part ofthe —C(═N—Z¹—R¹)—NH—Z²—R² motif may be part of a hetercyclic ring, whilethe other part of the motif has the meaning defined for Z¹, Z², R¹ andR², respectively. In some interesting embodiments, —C═N—Z¹—R¹ may formpart of a heterocyclic ring selected from the group consisting of a1,2-diazole ring, an isoxazole ring, a 1,2,4-triazole ring, and a1,2,4-oxadiazole ring, or -C-NH-Z²—R² may form part of a heterocyclicring selected from the group consisting of a 1,2-diazoline ring, anisoxazoline ring, a 1,2,4-triazoline ring, and a 1,2,4-oxadiazolinering. Such heterocyclic rings may be substituted as described above.

In some embodiments, at least one of R¹ and R² is hydrogen, e.g. bothare hydrogen. Further, in some embodiment, which may be combined withthe embodiments mentioned before, at least one of Z¹ and Z² is a singlebond, e.g. both are a single bond. In special embodiments, R¹ and R² areboth hydrogen, and Z¹ and Z² are both a single bond.

It is believed that the —C(═N—Z¹—R¹)—NH—Z²—R² motif is particularlyimportant for the stabilizing effect of the stabilizing agent (iii). Inparticular, it is believed that the —C(═N—Z¹—R¹)—NH—Z²—R² motif mimicsan arginine moiety of a substrate for the Factor VII polypeptide.

In more specific embodiments, the stabilizing agent (iii) is at leastone selected from the group consisting of amidine compounds comprising a—C—C(═N—Z¹—R¹)—NH—Z²—R² motif and guanidines compounds comprising a>N—C(═N—Z¹—R¹)—NH—Z²—R² motif.

In some embodiments, the stabilizing agent (iii) is at least one amidinecompound selected from the group consisting of benzamidines comprisingthe motif —C₆H₄—C(═N—Z¹—R¹)—NH—Z²—R², wherein C₆H₄ denotes an optionallysubstituted benzene ring, of which benzamidine (R¹ and R² are hydrogenand Z¹ and Z² are a single bond) constitutes a particular embodiment(see the Experimental section).

In other particular embodiments thereof, the benzamidines comprises themotif >N—C₆H₄—C(═N—Z¹—R¹)—NH—Z²—R², wherein C₆H₄ denotes an optionallysubstituted benzene ring, i.e. an o-amino-benzamidine, am-amino-benzamidine or a p-amino-benzamidine, of which ap-amino-benzamidine is the currently most preferred.

Further illustrative examples of p-amino-benzamidines are thosedisclosed by Aventis in EP 1 162 194 A1, cf. in particular those definedin claims 1-6 and in sections [0009]-[0052], and in EP 1 270 551 A1, cf.in particular claims 1 and 2 and sections [0010]-[0032].

In another embodiment, the stabilizing agent (iii) is at least oneguanidine compound selected from the group consisting of guanidinescompounds comprising a —CH₂—NH—C(═N—Z¹—R¹)—NH—Z²—R² motif. Examples ofguanidine compounds are those selected from the group consisting ofarginine, arginine derivatives and peptides of 2-5 amino acid residuescomprising at least one arginine residue. Arginine constitutes aparticular embodiment (see the Experimental section).

The term “arginine derivatives” is intended to encompass argininehomologues, N-terminal functionalised arginines (e.g. N-methylated andN-acylated (e.g. acetylated) derivatives), C-terminal functionalisedarginines (e.g. C-amidated, C-alkylamidated, and C-alkylatedderivatives), and combinations thereof.

As mentioned above, the one crucial motif of the stabilizing agents is—C(═N—Z¹—R¹)—NH—Z²—R². Other parts of the stabilizing agent may also beimportant, in particular with respect to optimization of the stabilizingeffect and the tolerance by the patient. Typically, the stabilizingagent has the formula Y—C(═N—Z¹—R¹)—NH—Z²—R², wherein Y is an organicradical. The radical Y is typically selected in order to improve theefficiency of the stabilizing effect. Also, the radical Y may compriseone or more further motifs of the formula —C(═N—Z¹—R¹)—NH—Z²—R².

The molecular weight of the stabilizing agent is typically at the most1000 Da, such as at the most 500 Da.

The compounds of the present invention may have one or more asymmetriccentres and unless otherwise indicated it is intended that stereoisomers(optical isomers), as separated, pure or partially purifiedstereoisomers or racemic mixtures thereof are included in the scope ofthe invention.

The concentration of the stabilizing agent (or agents) (iii) istypically at least 1 μM. The desirable (or necessary) concentrationtypically depends on the selected stabilizing agent (or agents), morespecifically on the binding affinity of the selected stabilizing agentto the Factor VII polypeptide.

In different embodiments, the stabilizing agent (iii) is present in aconcentration of at least 5 μM, at least 10 μM, at least 20 μM, at least50 μM, at least 100 μM, at least 150 μM, at least 250 μM, at least 500μM, at least 1 mM, at least 2 mM, at least 4 mM, at least 5 mM, at least8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20 mM,such as, e.g., in the range of 1-10000 μM, 10-10000 μM, 20-10000 μM,50-10000 μM, 10-5000 μM, 10-2000 μM, 20-5000 μM, 20-2000 μM, 50-5000 μM,0.1-100 mM, 0.1-75 mM, 0.1-50 mM, 0.1-10 mM, 0.2-75 mM, 0.2-50 mM,0.2-20 mM, 0.5-75 mM, or 0.5-50 mM.

In one embodiment, the stabilizing agent (iii) is benzamidine and theconcentration of said agent is at least 1 mM, such as, e.g., at least 2mM, although it is envisaged that substituted benzamidines may be morepotent for what reason they can be added in lower concentrations.

In one embodiment, the stabilizing agent (iii) is not benzamidine.

In one embodiment, the stabilizing agent (iii) is arginine and theconcentration of said agent is at least 10 mM, such as, e.g., at least50 mM.

In another embodiment, the stabilizing agent (iii) isp-amino-benzamidine and the concentration of said agent is at least0.001 mM

In another embodiment, the stabilizing agent (iii) is2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[(S)-1-(3-methoxyphenyl)-ethyl]-acetamidewith the formula

and the concentration of said agent is at least 0.001 mM.

In another embodiment, the stabilizing agent (iii) is2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[(S)-1-phenylethyl]-aetamide.with the formula

and the concentration of said agent is at least 0.001 mM.

In another embodiment, the stabilizing agent (iii) isN-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide withthe formula

and the concentration of said agent is at least 0.001 mM.

In various embodiments, the molar ratio between the stabilizing agent(iii) and FVII polypeptide (agent (iii):FVII) is: above 0.1, above 0.5,above 1, above 2, above 5, above 10, above 25, above 100, above 250,above 1000, above 2500, or above 5000, such as, e.g., in the range of0.1-10000, 0.1-5000, 0.1-2500, 0.1-1000, 0.1-250, 0.1-100, 0.1-25,0.1-10, 0.5-10000, 0.5-5000, 0.5-2500, 0.5-1000, 0.5-250, 0.5-100,0.5-25, 0.5-10, 1-10000, 1-5000, 1-2500, 1-1000, 1-250, 1-100; 1-25;1-10, 10-10000, 10-5000, 10-250, 1000-10000, or 1000-5000.

The desirable concentration typically depends on the selectedstabilizing agent (or agents), more specifically on the binding affinityof the selected agent to the Factor VII polypeptide.

The biological effect of the pharmaceutical composition is mainlyascribed to the presence of the Factor VII polypeptide, although otheractive ingredients may be included in combination with the Factor VIIpolypeptide.

As used herein, the term “Factor VII polypeptide” encompasses wild-typeFactor VII (i.e. a polypeptide having the amino acid sequence disclosedin U.S. Pat. No. 4,784,950), as well as variants of Factor VIIexhibiting substantially the same or improved biological activityrelative to wild-type Factor VII. The term “Factor VII” is intended toencompass Factor VII polypeptides in their uncleaved (zymogen) form, aswell as those that have been proteolytically processed to yield theirrespective bioactive forms, which may be designated Factor VIIa.Typically, Factor VII is cleaved between residues 152 and 153 to yieldFactor VIIa. The term “Factor VII polypeptide” also encompassespolypeptides, including variants, in which the Factor VIIa biologicalactivity has been substantially modified or somewhat reduced relative tothe activity of wild-type Factor VIIa. These polypeptides include,without limitation, Factor VII or Factor VIIa into which specific aminoacid sequence alterations have been introduced that modify or disruptthe bioactivity of the polypeptide.

The biological activity of Factor VIIa in blood clotting derives fromits ability to (i) bind to Tissue Factor (TF) and (ii) catalyze theproteolytic cleavage of Factor IX or Factor X to produce activatedFactor IX or X (Factor IXa or Xa, respectively).

For the purposes of the invention, biological activity of Factor VIIpolypeptides (“Factor VII biological activity”) may be quantified bymeasuring the ability of a preparation to promote blood clotting, cf.Assay 4 described herein. In this assay, biological activity isexpressed as the reduction in clotting time relative to a control sampleand is converted to “Factor VII units” by comparison with a pooled humanserum standard containing 1 unit/mL Factor VII activity. Alternatively,Factor VIIa biological activity may be quantified by (i) measuring theability of Factor VIIa or a Factor VII-related polypeptide to produceactivated Factor X (Factor Xa) in a system comprising TF embedded in alipid membrane and Factor X. (Persson et al., J. Biol. Chem.272:19919-19924, 1997); (ii) measuring Factor X hydrolysis in an aqueoussystem (“In Vitro Proteolysis Assay”, see Assay 2 below); (iii)measuring the physical binding of Factor VIIa or a Factor VII-relatedpolypeptide to TF using an instrument based on surface plasmon resonance(Persson, FEBS Letts. 413:359-363, 1997); (iv) measuring hydrolysis of asynthetic substrate by Factor VIIa and/or a Factor VII-relatedpolypeptide (“In Vitro Hydrolysis Assay”, see Assay 1 below); or (v)measuring generation of thrombin in a TF-independent in vitro system(see Assay 3 below).

Factor VII variants having substantially the same or improved biologicalactivity relative to wild-type Factor VIIa encompass those that exhibitat least about 25%, such as, e.g., at least about 50%, at least about75% or at least about 90% of the specific activity of Factor VIIa thathas been produced in the same cell type, when tested in one or more of aclotting assay (Assay 4), proteolysis assay (Assay 2), or TF bindingassay as described above. Factor VII variants having substantiallyreduced biological activity relative to wild-type Factor VIIa are thosethat exhibit less than about 25%, such as, e.g., less than about 10%, orless than about 5% of the specific activity of wild-type Factor VIIathat has been produced in the same cell type when tested in one or moreof a clotting assay (Assay 4), proteolysis assay (Assay 2), or TFbinding assay as described above. Factor VII variants having asubstantially modified biological activity relative to wild-type FactorVII include, without limitation, Factor VII variants that exhibitTF-independent Factor X proteolytic activity and those that bind TF butdo not cleave Factor X.

Variants of Factor VII, whether exhibiting substantially the same orbetter bioactivity than wild-type Factor VII, or, alternatively,exhibiting substantially modified or reduced bioactivity relative towild-type Factor VII, include, without limitation, polypeptides havingan amino acid sequence that differs from the sequence of wild-typeFactor VII by insertion, deletion, or substitution of one or more aminoacids.

Non-limiting examples of Factor VII variants having substantially thesame biological activity as wild-type Factor VII include S52A-FVIIa,S60A-FVIIa (Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998);FVIIa variants exhibiting increased proteolytic stability as disclosedin U.S. Pat. No. 5,580,560; Factor VIIa that has been proteolyticallycleaved between residues 290 and 291 or between residues 315 and 316(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); oxidized formsof Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54,1999); FVII variants as disclosed in PCT/DK02/00189; and FVII variantsexhibiting increased proteolytic stability as disclosed in WO 02/38162(Scripps Research Institute); FVII variants having a modified Gla-domainand exhibiting an enhanced membrane binding as disclosed in WO 99/20767(University of Minnesota); and FVII variants as disclosed in WO 01/58935(Maxygen ApS).

Non-limiting examples of Factor VII variants having increased biologicalactivity compared to wild-type FVIIa include FVII variants as disclosedin WO 01/83725, WO 02/22776, WO 02/077218, WO 03/27147, WO 03/37932; WO02/38162 (Scripps Research Institute); and FVIIa variants with enhancedactivity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic ResInst.).

Non-limiting examples of Factor VII variants having substantiallyreduced or modified biological activity relative to wild-type Factor VIIinclude R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990).

Examples of Factor VII polypeptides include, without limitation,wild-type Factor VII, L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII,L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII,K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII,V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII,V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII,E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and 5336G-FVII,L305V/K337A-FVII, L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII,L305V/V158T-FVII, L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII,L305V/K337A/E296V-FVII, L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII,L305V/V158D/E296V-FVII, L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII,L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII,L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII,L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII,L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII,L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII, S314E/K316Q-FVII,S314E/L305V-FVII, S314E/K337A-FVII, S314E/V158D-FVII, S314E/E296V-FVII,S314E/M298Q-FVII, S314E/V158T-FVII, K316H/L305V-FVII, K316H/K337A-FVII,K316H/V158D-FVII, K316H/E296V-FVII, K316H/M298Q-FVII, K316H/V158T-FVII,K316Q/L305V-FVII, K316Q/K337A-FVII, K316Q/V158D-FVII, K316Q/E296V-FVII,K316Q/M298Q-FVII, K316Q/V158T-FVII, S314E/L305V/K337A-FVII,S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII, S314E/L305V/M298Q-FVII,S314E/L305V/V158T-FVII, S314E/L305V/K337A/V158T-FVII,S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296V-FVII,S314E/L305V/K337A/V158D-FVII, S314E/L305V/V158D/M298Q-FVII,S314E/L305V/V158D/E296V-FVII, S314E/L305V/V158T/M298Q-FVII,S314E/L305V/V158T/E296V-FVII, S314E/L305V/E296V/M298Q-FVII,S314E/L305V/V158D/E296V/M298Q-FVII, S314E/L305V/V158T/E296V/M298Q-FVII,S314E/L305V/V158T/K337A/M298Q-FVII, S314E/L305V/V158T/E296V/K337A-FVII,S314E/L305V/V158D/K337A/M298Q-FVII, S314E/L305V/V158D/E296V/K337A-FVII,S314E/L305V/V158D/E296V/M298Q/K337A-FVII,S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII,K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII,K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII,K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII,K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII,K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII,K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII,K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII,K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A-FVII,K316H/L305V/V158D/E296V/M298Q/K337A-FVII,K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII,K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII,K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII,K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII,K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII,K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII,K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII,K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII,K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A -FVII,K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII,F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII,F374Y/L305V/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII,F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII,F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII,F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII,F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII,F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII,F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII,F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII,F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII,F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII,F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII,F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII,F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII,F374Y/K337A/V158T/E296V-FVII, F374Y/K337A/M298Q/E296V-FVII,F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII,F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII,F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII,F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-FVII,F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A -FVII, F374Y/L305V/E296V/M298Q/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII,F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII,F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII,F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII,F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII,F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII,F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII,F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII,F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII,F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,F374Y/L305V/E296V/K337A/V158T/S314E-FVII,F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,F374Y/L305V/V158D/E296V/K337A/S314E-FVII,F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Factor VIIa lackingthe Gla domain; and P11Q/K33E-FVII, T106N-FVII, K143N/N145T-FVII,V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII,K143N/N145T/R315N/V317T-FVII; and FVII having substitutions, additionsor deletions in the amino acid sequence from 233Thr to 240Asn, FVIIhaving substitutions, additions or deletions in the amino acid sequencefrom 304Arg to 329Cys, and FVII having substitutions, deletions, oradditions in the amino acid sequence Ile153-Arg223.

In some embodiments, the Factor VII polypeptide is human Factor VIIa(hFVIIa), preferably recombinantly made human Factor VIIa (rhVIIa).

In other embodiments, the Factor VII polypeptide is a Factor VIIsequence variant.

In some embodiments, the Factor VII polypeptide has a glycosylationdifferent from wild-type human Factor VII.

In various embodiments, e.g. those where the Factor VII polypeptide is aFactor VII-related polypeptide or a Factor VII sequence variant, theratio between the activity of the Factor VII polypeptide and theactivity of native human Factor VIIa (wild-type FVIIa) is at least about1.25, preferably at least about 2.0, or 4.0, most preferred at leastabout 8.0, when tested in the “In Vitro Proteolysis Assay” (Assay 2) asdescribed in the present specification.

In some embodiments, the Factor VII polypeptides are Factor VII-relatedpolypeptides, in particular variants, wherein the ratio between theactivity of said Factor VII polypeptide and the activity of native humanFactor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the“In Vitro Hydrolysis Assay” (see Assay 1 below); in other embodiments,the ratio is at least about 2.0; in further embodiments, the ratio is atleast about 4.0.

In a pharmaceutical composition, it is often desirable that theconcentration of the active ingredient is such that the application of aunit dose does not cause unnecessary discomfort to the patient. Thus, aunit dose of more than about 2-10 mL is often undesirable. For thepurpose of the present invention, the concentration of the Factor VIIpolypeptide is therefore at least 0.01 mg/mL. In different embodiments,the Factor VII polypeptide is present in a concentration of 0.01-20mg/mL; 0.1-20 mg/mL; 0.1-15 mg/mL; 0.1-10 mg/mL; 0.5-5.0 mg/mL; 0.6-4.0mg/mL; 1.0-4.0 mg/mL; 0.1-5 mg/mL; 0.1-4.0 mg/mL; 0.1-2 mg/mL; or0.1-1.5 mg/mL.

Factor VIIa concentration is conveniently expressed as mg/mL or asIU/mL, with 1 mg usually representing 43,000-56,000 IU or more.

In order to render the liquid, aqueous pharmaceutical composition usefulfor direct parenteral administration to a mammal such as a human, it isnormally required that the pH value of the composition is held withincertain limits, such as from about 4.0 to about 9.0. To ensure asuitable pH value under the conditions given, the pharmaceuticalcomposition also comprises a buffering agent (ii) suitable for keepingpH in the range of from about 4.0 to about 9.0.

The term “buffering agent” includes those agents or combinations ofagents that maintain the solution pH in an acceptable range from about4.0 to about 9.0.

In one embodiment, the buffering agent (ii) is at least one componentselected from the groups consisting of acids and salts of MES, PIPES,ACES, BES, TES, HEPES, TRIS, histidine (e.g. L-histidine), imidazole,glycine, glycylglycine, glycinamide, phosphoric acid (e.g. sodium orpotassium phosphate), acetic acid (e.g. ammonium, sodium or calciumacetate), lactic acid, glutaric acid, citric acid (e.g. sodium orpotassium citrate), tartaric acid, malic acid, maleic acid, and succinicacid. It should be understood that the buffering agent may comprise amixture of two or more components, wherein the mixture is able toprovide a pH value in the specified range. As examples can be mentionedacetic acid and sodium acetate, etc.

The concentration of the buffering agent is chosen so as to maintain thepreferred pH of the solution. In various embodiments, the concentrationof the buffering agent is 1-100 mM; 1-50 mM; 1-25 mM; or 2-20 mM.

In one embodiment, the pH of the composition is kept from about 4.0 toabout 9.0; from 5.0 to about 9.0; from about 5.0 to about 8.0; such asfrom about 5.0 to about 7.5; from about 5.0 and about 7.0; from about5.0 to about 6.5; from about 5.0 to about 6.0; from about 5.5 to about7.0; from about 5.5 to about 6.5; from about 6.0 to about 7.0; fromabout 6.0 to about 6.5; from about 6.3 to about 6.7, or from about 5.2to about 5.7.

In addition to the three mandatory components, the liquid, aqueouspharmaceutical composition may comprise additional components beneficialfor the preparation, formulation, stability, or administration of thecomposition.

Hence, the pharmaceutical composition may also include a non-ionicsurfactant. Surfactants (also known as detergents) generally includethose agents which protect the protein from air/solution interfaceinduced stresses and solution/surface induced stresses (e.g. resultingin protein aggregation).

Typical types of non-ionic surfactants are polysorbates, poloxamers,polyoxyethylene alkyl ethers, polyethylene/polypropylene blockco-polymers, polyethyleneglycol (PEG), polyxyethylene stearates, andpolyoxyethylene castor oils.

Illustrative examples of non-ionic surfactants are Tween®, polysorbate20, polysorbate 80, Brij-35 (polyoxyethylene dodecyl ether), poloxamer188, poloxamer 407, PEG8000, Pluronic® polyols, polyoxy-23-lauryl ether,Myrj 49, and Cremophor A.

In one embodiment, the non-ionic surfactant is present in an amount of0.005-2.0% by weight.

Also, the composition may further comprise a tonicity modifying agent(v).

As used herein, the term “tonicity modifying agent” includes agentswhich contribute to the osmolality of the solution. The tonicitymodifying agent (v) includes at least one agent selected from the groupconsisting of neutral salts, amino acids, peptides of 2-5 amino acidresidues, monosaccharides, disaccharides, polysaccharides, and sugaralcohols. In some embodiments, the composition comprises two or more ofsuch agents in combination.

By “neutral salt” is meant a salt that is neither an acid nor a basewhen dissolved in an aqueous solution.

In one embodiment, at least one tonicity modifying agent (v) is aneutral salt selected from the groups consisting of sodium salts,potassium salts, calcium salts, and magnesium salts, such as sodiumchloride, potassium chloride, calcium chloride, calcium acetate, calciumgluconate, calcium laevulate, magnesium chloride, magnesium acetate,magnesium gluconate, and magnesium laevulate.

In a further embodiment, the tonicity modifying agent (v) includessodium chloride in combination with at least one selected from thegroups consisting of calcium chloride, calcium acetate, magnesiumchloride and magnesium acetate.

In a still further embodiment, the tonicity modifying agent (v) is atleast one selected from the group consisting of sodium chloride, calciumchloride, sucrose, glucose, and mannitol.

In different embodiments, the tonicity modifying agent (v) is present ina concentration of at least 1 mM, at least 5 mM, at least 10 mM, atleast 20 mM, at least 50 mM, at least 100 mM, at least 200 mM, at least400 mM, at least 800 mM, at least 1000 mM, at least 1200 mM, at least1500 mM, at least 1800 mM, at least 2000 mM, or at least 2200 mM.

In one series of embodiments, the tonicity modifying agent (v) ispresent in a concentration of 5-2200 mM, such as 25-2200 mM, 50-2200 mM,100-2200 mM, 200-2200 mM, 400-2200 mM, 600-2200 mM, 800-2200 mM,1000-2200 mM, 1200-2200 mM, 1400-2200 mM, 1600-2200 mM, 1800-2200 mM, or2000-2200 mM; 5-1800 mM, 25-1800 mM, 50-1800 mM, 100-1800 mM, 200-1800mM, 400-1800 mM, 600-1800 mM, 800-1800 mM, 1000-1800 mM, 1200-1800 mM,1400-1800 mM, 1600-1800 mM; 5-1500 mM, 25-1400 mM, 50-1500 mM, 100-1500mM, 200-1500 mM, 400-1500 mM, 600-1500 mM, 800-1500 mM, 1000-1500 mM,1200-1500 mM; 5-1200 mM, 25-1200 mM, 50-1200 mM, 100-1200 mM, 200-1200mM, 400-1200 mM, 600-1200 mM, or 800-1200 mM.

In one embodiment of the invention, at least one tonicity modifyingagent (v) is an ionic strength modifying agent (v/a).

As used herein, the term “ionic strength modifying agent” includesagents which contribute to the ionic strength of the solution. Theagents include, but are not limited to, neutral salts, amino acids,peptides of 2 to 5 amino acid residues. In some embodiments, thecomposition comprises two or more of such agents in combination.

Non-limiting examples of ionic strength modifying agents (v/a) areneutral salts such as sodium chloride, potassium chloride, calciumchloride and magnesium chloride. In one embodiment, the agent (v/a) issodium chloride.

The term “ionic strength” is the ionic strength of the solution (μ)which is defined by the equation: μ=½Σ([i](Z_(i) ²)), where μ is theionic strength, [i] is the millimolar concentration of an ion, and Z_(i)is the charge (+ or −) of that ion “(see, e.g., Solomon, Journal ofChemical Education, 78(12):1691-92, 2001; James Fritz and George Schenk:Quantitative Analytical Chemistry, 1979).

In different embodiments of the invention, the ionic strength of thecomposition is at least 50 mM, such as at least 75 mM, at least 100 mM,at least 150 mM, at least 200 mM, at least 250 mM, at least 400 mM, atleast 500 mM, at least 650 mM, at least 800 mM, at least 1000 mM, atleast 1200 mM, at least 1600 mM, at least 2000 mM, at least 2400 mM, atleast 2800 mM, or at least 3200 mM.

In some specific embodiments, the total concentration of the tonicitymodifying agent (v) and the ionic strength modifying agent (v/a) is inthe range of 1-1000 mM, such as 1-500 mM, 1-300 mM, 10-200 mM, or 20-150mM; or such as 100-1000 mM, 200-800 mM, or 500-800 mM, depending on theeffect any other ingredients may have on the tonicity and ionicstrength.

In one embodiment, the composition is isotonic; in another, it ishypertonic.

The term “isotonic” means “isotonic with serum”, i.e. at about 300±50milliosmol/kg. The tonicity is meant to be a measure of osmolality ofthe solution prior to administration. The term “hypertonic” is meant todesignate levels of osmolality above the physiological level of serum,such as levels above 300±50 milliosmol/kg.

Also, a particular embodiment of the present invention relates to thecombination of the stabilizing agent (iii) with a fairly highconcentration of an ionic strength modifying agent (v/a). In oneembodiment thereof, the ionic strength modifying agent (v/a) is selectedfrom the group consisting of sodium salts, calcium salts and magnesiumsalts. In this embodiment, the ionic strength modifying agent (v/a),i.e. the sodium salt, calcium salt and/or magnesium salt, is present ina concentration of 15-1500 mM, such as 15-1000 mM, 25-1000 mM, 50-1000mM, 100-1000 mM, 200-1000 mM, 300-1000 mM, 400-1000 mM, 500-1000 mM,600-1000 mM, 700-1000 mM; 15-800 mM, 25-800 mM, 50-800 mM, 100-800 mM,200-800 mM, 300-800 mM, 400-800 mM, 500-800 mM; 15-600 mM, 25-600 mM,50-600 mM, 100-600 mM, 200-600 mM, 300-600 mM; 15-400 mM, 25-400 mM,50-400 mM, or 100-400 mM.

Within these embodiments, sodium salt may be sodium chloride, thecalcium salt may be selected from the group consisting of calciumchloride, calcium acetate, calcium gluconate, and calcium laevulate, andthe magnesium salt may be selected from the group consisting ofmagnesium chloride, magnesium acetate, magnesium gluconate, magnesiumlaevulate, and magnesium salts of strong acids. In a more specificembodiment, a calcium salt and/or a magnesium salt is/are used incombination with sodium chloride.

In one embodiment, the composition comprises one or more ionic strengthmodifying agents selected from the group consisting of calcium (Ca²⁺)salts and magnesium (Mg²⁺) salts, e.g. one or more salts selected fromthe group consisting of calcium chloride, calcium acetate, calciumgluconate, calcium laevulate, magnesium chloride, magnesium acetate,magnesium sulphate, magnesium gluconate, magnesium laevulate, magnesiumsalts of strong acids.

In one embodiment, the Calcium (Ca2+) and/or Magnesium (Mg2+) is presentin a concentration of at least about 0.1 μM, such as, e.g., at leastabout 0.5 μM, at least about 1 μM, at least about 5 μM, at least about10 μM, at least about 50 μM, at least about 100 μM, at least about 1 mM,at least about 2 mM, at least about 5 mM, or at least about 10mM. In aparticular embodiment the composition comprises at least 2 mM Ca2+.

In various embodiments, the molar ratio between calcium (Ca2+) and/ormagnesium ions (Mg2+) and FVII polypeptide is: 0.001-750; 0.001-250;0.001-100; 0.001-10; 0.001-1.0; 0.001-0.5; 0.5-750; 0.5-250; 0.5-100;0.5-10; 0.5-1.0; 0.001-0.4999; 0.005-0.050.

In one embodiment of the present invention, the molar ratio ofnon-complexed calcium (Ca2+) and/or magnesium (Mg2+) to the Factor VIIpolypeptide is lower than 0.5, e.g. in the range of 0.001-0.499, such as0.005-0.050, or in the range of 0.000-0.499, such as in the range of0.000-0.050, or about 0.000. In one embodiment of the present invention,the molar ratio of non-complexed calcium (Ca2+) to the Factor VIIpolypeptide is lower than 0.5, e.g. in the range of 0.001-0.499, such as0.005-0.050, or in the range of 0.000-0.499, such as in the range of0.000-0.050, or about 0.000.

When used herein, the term “the concentration of non-complexed calciumand/or magnesium ions” is intended to mean the difference between thetotal concentration of calcium and/or magnesium ions and theconcentration of calcium and/or magnesium bound to calcium/magnesiumchelators. In this regard, the Factor VII polypeptide is not regarded asa “calcium/magnesium chelator” although calcium and/or magnesium isexpected to bind to, or become associated with, the Factor VIIpolypeptide under certain conditions.

In another embodiment, the molar ratio of non-complexed calcium and/ormagnesium ions to the Factor VII polypeptide is above 0.5. In anotherembodiment, the molar ratio of non-complexed calcium ions to the FactorVII polypeptide is above 0.5.

In order to obtain the low relative ratio between calcium and/ormagnesium ions (Ca2+) and the Factor VII polypeptide, it may benecessary or desirable to remove excess calcium and/or magnesium ions,e.g., by contacting the composition with an ion-exchange material underconditions suitable for removing Ca2+ and/or Mg2+, or to add a calcium/magnesium chelator in order to bind (complex) excess calcium and/ormagnesium ions. This is particularly relevant where the ratio betweencalcium and/or magnesium ions and the Factor VII polypeptide in asolution from a process step preceding the formulation step exceeds thelimit stated above. Examples of “calcium/magnesium chelators” includeEDTA, citric acid, NTA, DTPA, tartaric acid, lactic acid, malic acid,succinic acid, HIMDA, ADA and similar compounds.

In a further embodiment, the composition further comprises anantioxidant (vi). In different embodiments, the antioxidant is selectedfrom the group consisting of L-methionine, D-methionine, methionineanalogues, methionine-containing peptides, methionine-homologues,ascorbic acid, cysteine, homocysteine, gluthatione, cystine, andcysstathionine. In a preferred embodiment, the antioxidant isL-methionine.

The concentration of the antioxidant is typically 0.1-5.0 mg/mL, such as0.1-4.0 mg/mL, 0.1-3.0 mg/mL, 0.1-2.0 mg/ml, or 0.5-2.0 mg/mL.

In particular embodiments, the composition does not include anantioxidant; instead the susceptibility of the Factor VII polypeptide tooxidation is controlled by exclusion of atmospheric air. The use of anantioxidant may of course also be combined with the exclusion ofatmospheric air.

Thus, the present invention also provides an air-tight container (e.g. avial or a cartridge (such as a cartridge for a pen applicator))containing a liquid, aqueous pharmaceutical composition as definedherein, and optionally an inert gas.

The inert gas may be selected from the groups consisting of nitrogen,argon, etc. The container (e.g. vial or cartridge) is typically made ofglass or plastic, in particular glass, optionally closed by a rubberseptum or other closure means allowing for penetration with preservationof the integrity of the pharmaceutical composition. In a particularembodiment hereof, the composition does not comprise a preservative(vii). In a further embodiment, the container is a vial or cartridgeenclosed in a sealed bag, e.g. a sealed plastic bag, such as a laminated(e.g. metal (such as aluminium) laminated plastic bag).

In addition to the mandatory components, the non-ionic surfactant (iv),the tonicity modifying agent (v) and the optional antioxidant (vi), thepharmaceutical composition may further comprise a preservative (vii).

A preservative may be included in the composition to retard microbialgrowth and thereby allow “multiple use” packaging of the Factor VIIpolypeptides. Examples of preservatives include phenol, benzyl alcohol,orto-cresol, meta-cresol, para-cresol, methyl paraben, propyl paraben,benzalkonium chloride, and benzethonium chloride. The preservative isnormally included at a concentration of 0.1-20 mg/mL depending on the pHrange and type of preservative.

Still further, the composition may also include one or more agentscapable of inhibiting deamidation and isomerisation.

In one embodiment, the liquid, aqueous pharmaceutical compositioncomprises:

0.1-20 mg/mL of a Factor VII polypeptide (i);

a buffering agent (ii) suitable for keeping pH in the range of fromabout 4.0 to about 9.0;

at least one stabilizing agent (iii) comprising the motif—C₆H₄—C(═N—Z¹—R¹)—NH—Z²—R² in a concentration of at least 5 μM;

a non-ionic surfactant (iv); and

at least one tonicity modifying agent (v) in a concentration of at least5 mM.

In another embodiment, the liquid, aqueous pharmaceutical compositioncomprises: 0.1-10 mg/mL of a Factor VII polypeptide (i);

a buffering agent (ii) suitable for keeping pH in the range of fromabout 4.0 to about 9.0;

at least one stabilizing agent (iii) comprising the motif—CH₂—NH—C(═N—Z¹—R¹)—NH—Z²—R² in a concentration of at least 500 μM;

a non-ionic surfactant (iv); and

at least one tonicity modifying agent (v) in a concentration of at least5 mM.

As used herein, pH values specified as “about” are understood to be±0.1, e.g. about pH 8.0 includes pH 8.0±0.1.

Percentages are (weight/weight) both when referring to solids dissolvedin solution and liquids mixed into solutions. For example, for Tween®,it is the weight of 100% stock/weight of solution.

The compositions according to the present invention are useful as stableand preferably ready-to-use compositions of Factor VII polypeptides.Furthermore, it is believed that the principles, guidelines and specificembodiments given herein are equally applicable for bulk storage ofFactor VII polypeptides, mutatis mutandis. The compositions aretypically stable for at least six months, and preferably up to 36months; when stored at temperatures ranging from 2° C. to 8° C. Thecompositions are chemically and/or physically stable, in particularchemically stable, when stored for at least 6 months at from 2° C. to 8°C.

The term “Stable” is intended to denote that (i) after storage for 6months at 2° C. to 8° C. the composition retains at least 50% of itsinitial biological activity as measured by a one-stage clot assayessentially as described in Assay 4 of the present specification, or(ii) after storage for 6 months at 2° C. to 8° C., the increase incontent of heavy chain degradation products is at the most 40% (w/w) ofthe initial content of Factor VII polypeptide.

The term “initial content” relates to the amount of Factor VIIpolypeptides added to a composition upon preparation of the composition.

In one embodiment, the stable composition retains at least 70%, such as,e.g., at least 80%, at least 85%, at least 90%, or at least 95%, of itsinitial biological activity after storage for 6 months at 2 to 8° C.

In different embodiments of the invention, the stable compositionfurther retains at least 50% of its initial biological activity asmeasured by a one-stage clot assay essentially as described in Assay 4of the present specification after storage for at least 30 days, such as60 days or 90 days.

In various embodiments the increase in content of heavy chaindegradation products in the stable compositions is not more than about30% (w/w), not more than about 25% (w/w), not more than about 20% (w/w),not more than about 15% (w/w), not more than about 10% (w/w), not morethan about 5% (w/w), or not more than about 3% (w/w) of the initialcontent of Factor VII polypeptide.

For the purpose of determining the content of heavy chain degradationproducts, a reverse phase HPLC was run on a proprietary 4.5×250 mmbutyl-bonded silica column with a particle size of 5 μm and pore size300 Å. Column temperature: 70° C. A-buffer: 0.1% v/v trifluoraceticacid. B-buffer: 0.09% v/v trifluoracetic acid, 80% v/v acetonitrile. Thecolumn was eluted with a linear gradient from X to (X+13)% B in 30minutes. X was adjusted so that FVIIa elutes with a retention time ofapproximately 26 minutes. Flow rate: 1.0 mL/min. Detection: 214 nm.Load: 25 μg FVIIa.

The term “physical stability” of Factor VII polypeptides relates to theformation of insoluble and/or soluble aggregates in the form of dimeric,oligomeric and polymeric forms of Factor VII polypeptides as well as anystructural deformation and denaturation of the molecule. Physicallystable composition encompasses compositions which remains visuallyclear. Physical stability of the compositions is often evaluated bymeans of visual inspection and turbidity after storage of thecomposition at different temperatures for various time periods. Visualinspection of the compositions is performed in a sharp focused lightwith a dark background. A composition is classified as physicallyunstable, when it shows visual turbidity.

The term “chemical stability” is intended to relate to the formation ofany chemical change in the Factor VII polypeptides upon storage insolution at accelerated conditions. Examples are hydrolysis, deamidationand oxidation as well as enzymatic degradation resulting in formation offragments of Factor VII polypeptides. In particular, thesulphur-containing amino acids are prone to oxidation with the formationof the corresponding sulphoxides.

The term “chemically stable” is intended to designate a compositionwhich retains at least 50% of its initial biological activity afterstorage for 6 months at 2 to 8° C., as measured by a one-stage clotassay (Assay 4).

In a further aspect, the invention also provides a method for preparinga liquid, aqueous pharmaceutical composition of a Factor VIIpolypeptide, comprising the step of providing the

Factor VII polypeptide at a concentration of at least 0.01 mg/mL (i) ina solution comprising a buffering agent (ii) suitable for keeping pH inthe range of from about 4.0 to about 9.0; and at least one stabilizingagent (iii) comprising a —C(═N—Z¹—R¹)—NH—Z²—R² motif, wherein

Z¹ and Z² independently are selected from the group consisting of —O—,—S—, —NR^(H)— and a single bond, where R^(H) is selected from the groupconsisting of hydrogen, C₁₋₄-alkyl, aryl and arylmethyl, and R¹ and R²independently are selected from the group consisting of hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, optionally substituted heterocyclyl, or

Z² and R² are as defined above and —C═N—Z¹—R¹ forms part of aheterocyclic ring, or

Z¹ and R¹ are as defined above and —C—NH—Z²—R² forms part of aheterocyclic ring, or

—C(═N—Z¹—R¹)—NH—Z²—R² forms a hetercyclic ring wherein Z¹—R¹—R²—Z²— is abiradical.

Methods of Use

As will be understood, the liquid, aqueous pharmaceutical compositionsdefined herein can be used in the field of medicine. Thus, the presentinvention in particular provides the liquid, aqueous pharmaceuticalcompositions defined herein for use as a medicament, more particular foruse as a medicament for treating a Factor VII-responsive syndrome.

Consequently, the present invention also provides the use of the liquid,aqueous pharmaceutical composition as defined herein for the preparationof a medicament for treating a Factor VII-responsive syndrome, as wellas a method for treating a Factor VII-responsive syndrome, the methodcomprising administering to a subject in need thereof an effectiveamount of the liquid, aqueous pharmaceutical composition as definedherein.

The preparations of the present invention may be used to treat anyFactor VII-responsive syndrome, such as, e.g., bleeding disorders,including those caused by clotting Factor deficiencies (e.g., e.g.haemophilia A, haemophilia B, coagulation Factor XI deficiency,coagulation Factor VII deficiency); by thrombocytopenia or vonWillebrand's disease, or by clotting Factor inhibitors, and intracerebral haemorrhage, or excessive bleeding from any cause.

The preparations may also be administered to patients in associationwith surgery or other trauma or to patients receiving anticoagulanttherapy.

The term “effective amount” is the effective dose to be determined by aqualified practitioner, who may titrate dosages to achieve the desiredresponse. Factors for consideration of dose will include potency,bioavailability, desired pharmacokinetic/pharmacodynamic profiles,condition of treatment, patient-related factors (e.g. weight, health,age, etc.), presence of co-administered medications (e.g.,anticoagulants), time of administration, or other factors known to amedical practitioner.

The term “treatment” is defined as the management and care of a subject,e.g. a mammal, in particular a human, for the purpose of combating thedisease, condition, or disorder and includes the administration of aFactor VII polypeptide to prevent the onset of the symptoms orcomplications, or alleviating the symptoms or complications, oreliminating the disease, condition, or disorder. Pharmaceuticalcompositions according to the present invention containing a Factor VIIpolypeptide may be administered parenterally to subjects in need of sucha treatment. Parenteral administration may be performed by subcutaneous,intramuscular or intravenous injection by means of a syringe, optionallya pen-like syringe. Alternatively, parenteral administration can beperformed by means of an infusion pump.

In important embodiments, the pharmaceutical composition is adapted tosubcutaneous, intramuscular or intravenous injection according tomethods known in the art.

EXPERIMENTALS

General Methods

Assays Suitable for Determining Biological Activity of Factor VIIPolypeptides

Factor VII polypeptides useful in accordance with the present inventionmay be selected by suitable assays that can be performed as simplepreliminary in vitro tests. Thus, the present specification discloses asimple test (entitled “In Vitro Hydrolysis Assay”) for the activity ofFactor VII polypeptides.

In Vitro Hydrolysis Assay (Assay 1)

Native (wild-type) Factor VIIa and Factor VII polypeptide (bothhereinafter referred to as “Factor VIIa”) may be assayed for specificactivities. They may also be assayed in parallel to directly comparetheir specific activities. The assay is carried out in a microtiterplate (MaxiSorp, Nunc, Denmark). The chromogenic substrateD-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), finalconcentration 1 mM, is added to Factor VIIa (final concentration 100 nM)in 50 mM HEPES, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/mLbovine serum albumin. The absorbance at 405 nm is measured continuouslyin a SpectraMax™ 340 plate reader (Molecular Devices, USA). Theabsorbance developed during a 20-minute incubation, after subtraction ofthe absorbance in a blank well containing no enzyme, is used forcalculating the ratio between the activities of Factor VII polypeptideand wild-type Factor VIIa:Ratio=(A405 nm Factor VII polypeptide)/(A405 nm Factor VIIa wild-type).

Based thereon, Factor VII polypeptides with an activity lower than,comparable to, or higher than native Factor VIIa may be identified, suchas, for example, Factor VII polypeptides where the ratio between theactivity of the Factor VII polypeptide and the activity of native FactorVII (wild-type FVII) is about 1.0 versus above 1.0.

The activity of the Factor VII polypeptides may also be measured using aphysiological substrate such as Factor X (“In Vitro Proteolysis Assay”),suitably at a concentration of 100-1000 nM, where the Factor Xagenerated is measured after the addition of a suitable chromogenicsubstrate (eg. S-2765). In addition, the activity assay may be run atphysiological temperature.

In Vitro Proteolysis Assay (Assay 2)

Native (wild-type) Factor VIIa and Factor VII polypeptide (bothhereinafter referred to as “Factor VIIa”) are assayed in parallel todirectly compare their specific activities. The assay is carried out ina microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 μL 50 mM HEPES, pH 7.4, containing 0.1 MNaCl, 5 mM CaCl₂ and 1 mg/mL bovine serum albumin, are incubated for 15min. Factor X cleavage is then stopped by the addition of 50 μL 50 mMHEPES, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mL bovineserum albumin. The amount of Factor Xa generated is measured by theaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used for calculating the ratio between the proteolyticactivities of Factor VII polypeptide and wild-type Factor VIIa:Ratio=(A405 nm Factor VII polypeptide)/(A405 nm Factor VIIa wild-type).

Based thereon, Factor VII polypeptide with an activity lower than,comparable to, or higher than native Factor VIIa may be identified, suchas, for example, Factor VII polypeptides where the ratio between theactivity of the Factor VII polypeptide and the activity of native FactorVII (wild-type FVII) is about 1.0 versus above 1.0.

Thrombin Generation Assay (Assay 3)

The ability of Factor VIIa or Factor VII polypeptides to generatethrombin can also be measured in an assay (Assay 3) comprising allrelevant coagulation Factors and inhibitors at physiologicalconcentrations (minus Factor VIII when mimicking hemophilia Aconditions) and activated platelets (as described on p. 543 in Monroe etal. (1997) Brit. J. Haematol. 99, 542-547, which is hereby incorporatedherein as reference).

One-Stage Coagulation Assay (Clot Assay) (Assay 4)

Factor VII polypeptides may also be assayed for specific activities(“clot activity”) by using a a one-stage coagulation assay (Assay 4).For this purpose, the sample to be tested is diluted in 50 mMPIPES-buffer (pH 7.5), 0.1% BSA and 40 μl is incubated with 40 μl ofFactor VII deficient plasma and 80 μl of human recombinant tissue factorcontaining 10 mM Ca2+ and synthetic phospholipids. Coagulation times(clotting times) are measured and compared to a standard curve using areference standard in a parallel line assay.

Preparation and Purification of Factor VII Polypeptides

Human purified Factor VIIa suitable for use in the present invention ispreferably made by DNA recombinant technology, e.g. as described byHagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986, or asdescribed in European Patent No. 0 200 421 (ZymoGenetics, Inc.).

Factor VII may also be produced by the methods described by Broze andMajerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel,J. Clin. Invest. 71: 1836-1841, 1983. These methods yield Factor VIIwithout detectable amounts of other blood coagulation Factors. An evenfurther purified Factor VII preparation may be obtained by including anadditional gel filtration as the final purification step. Factor VII isthen converted into activated Factor VIIa by known means, e.g. byseveral different plasma proteins, such as Factor XIIa, IX a or Xa.Alternatively, as described by Bjoern et al. (Research Disclosure, 269September 1986, pp. 564-565), Factor VII may be activated by passing itthrough an ion-exchange chromatography column, such as Mono Q®(Pharmacia fine Chemicals) or the like, or by autoactivation insolution.

Factor VII-related polypeptides may be produced by modification ofwild-type Factor VII or by recombinant technology. Factor VII-relatedpolypeptides with altered amino acid sequence when compared to wild-typeFactor VII may be produced by modifying the nucleic acid sequenceencoding wild-type Factor VII either by altering the amino acid codonsor by removal of some of the amino acid codons in the nucleic acidencoding the natural Factor VII by known means, e.g. by site-specificmutagenesis.

It will be apparent to those skilled in the art that substitutions canbe made outside the regions critical to the function of the Factor VIIamolecule and still result in an active polypeptide. Amino acid residuesessential to the activity of the Factor VII polypeptide, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for coagulant, respectivelycross-linking activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-enzyme interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure that utilizes a super-coiled,double-stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemi-methylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art for creating, identifying andisolating variants may also be used, such as, for example, geneshuffling or phage display techniques.

Separation of polypeptides from their cell of origin may be achieved byany method known in the art, including, without limitation, removal ofcell culture medium containing the desired product from an adherent cellculture; centrifugation or filtration to remove non-adherent cells; andthe like.

Optionally, Factor VII polypeptides may be further purified.Purification may be achieved using any method known in the art,including, without limitation, affinity chromatography, such as, e.g.,on an anti-Factor VII antibody column (see, e.g., Wakabayashi et al., J.Biol. Chem. 261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988);hydrophobic interaction chromatography; ion-exchange chromatography;size exclusion chromatography; electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction and the like. See,generally, Scopes, Protein Purification, Springer-Verlag, New York,1982; and Protein Purification, J. C. Janson and Lars Ryden, editors,VCH Publishers, New York, 1989. Following purification, the preparationpreferably contains less than 10% by weight, more preferably less than5% and most preferably less than 1%, of non-Factor VII polypeptidesderived from the host cell.

Factor VII polypeptides may be activated by proteolytic cleavage, usingFactor XIIa or other proteases having trypsin-like specificity, such as,e.g., Factor IXa, kallikrein, Factor Xa, and thrombin. See, e.g.,Osterud et al., Biochem. 11:2853 (1972); Thomas, U.S. Pat. No.4,456,591; and Hedner et al., J. Clin. Invest. 71:1836 (1983).Alternatively, Factor VII polypeptides may be activated by passing itthrough an ion-exchange chromatography column, such as Mono Q®(Pharmacia) or the like, or by autoactivation in solution. The resultingactivated Factor VII polypeptide may then be formulated and administeredas described in the present application.

The following examples illustrate practice of the invention. Theseexamples are included for illustrative purposes only and are notintended in any way to limit the scope of the invention claimed.

WORKING EXAMPLES

In the below working examples the content of heavy chain degradationproducts is determined by RP-HPLC as described in the following:

Reverse phase HPLC was run on a proprietary 4.5×250 mm butyl-bondedsilica column with a particle size of 5 μm and pore size 300 Å. Columntemperature: 70° C. A-buffer: 0.1% v/v trifluoracetic acid. B-buffer:0.09% v/v trifluoracetic acid, 80% v/v acetonitrile. The column waseluted with a linear gradient from X to (X+13)% B in 30 minutes. X wasadjusted so that FVIIa elutes with a retention time of approximately 26minutes. Flow rate: 1.0 mL/min. Detection: 214 nm. Load: 25 μg FVIIa.

In the below examples the clot activity is measured using a one stageclot assay essentially as described in Assay 4 of the presentspecification.

Example 1

In order to investigate the effect of benzamidine on the stability ofrFVIIa the following formulations were prepared:

Formulation 1:

-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Sodium acetate-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 mM Benzamidine-   pH=6.5    Formulation 2:-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Sodium acetate-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 mM Benzamidine-   pH=7.0    Formulation 3:-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Sodium acetate-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=6.5    Formulation 4:-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Sodium acetate-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.0

The formulations were prepared by adding 10 mM histidine, 10 mM sodiumacetate and 50 mM benzamidine (only for formulations 1 and 2) to a 1.0mg/mL bulk solution of rFVIIa already containing glycylglycine, sodiumchloride and calcium chloride in the above mentioned concentrations. pHwas finally adjusted to 6.5 and 7.0, respectively, with 1 M sodiumhydroxide and 1 M hydrochloric acid.

The formulations were stored at a temperature of 5° C. and 30° C., andthe analyses for formation of Heavy chain degradation products wereperformed at time points shown in the table (Table 1).

TABLE 1 Formation of Heavy chain degradation (hcd) products inbenzamidine formulations % hcd % hcd % hcd % hcd ½ months 1 months 2months Formulation 0 months 30° C. 30° C. 5° C. 30° C. 5° C. 1 (pH 6.5)7.5 9.5 11.6 7.5 14.6 7.7 2 (pH 7.0) 7.3 12.4 17.2 8.0 23.5 8.6 3 (pH6.5) 8.1 — 17.7 16.3 22.7 — 4 (pH 7.0) 9.6 — 29.9 32.5 38.6 — % hcd %hcd % hcd 3 months 6 months 14 months Formulation 30° C. 5° C. 30° C. 5°C. 5° C. 1 (pH 6.5) 17.5 8.1 20.4 8.9 10.5 2 (pH 7.0) 28.4 9.4 31.5 11.515.4 3 (pH 6.5) — 30.2 25.7 42.1 — 4 (pH 7.0) — 56.9 45.5 67.3 —

As it can be seen from Table 1, after 6 months of storage at 5° C. theincrease in the content of Heavy chain degradation products in thereference formulations (3 and 4) was 34.0% and 57.7%, respectively,whereas the increase in the content of Heavy chain degradation productsin the illustrative compositions (1 and 2) was only 1.4% and 4.2%,respectively. After 14 months of storage at 5° C., the increase in thecontent of Heavy chain degradation products in the illustrativecompositions (1 and 2) was only 3.0% and 8.1%, respectively.

The content of heavy chain degradation products is determined by RP-HPLCas described in the following:

Reverse phase HPLC was run on a proprietary 4.5×250 mm butyl-bondedsilica column with a particle size of 5 μm and pore size 300 Å. Columntemperature: 70° C. A-buffer: 0.1% v/v trifluoracetic acid. B-buffer:0.09% v/v trifluoracetic acid, 80% v/v acetonitrile. The column waseluted with a linear gradient from X to (X+13)% B in 30 minutes. X wasadjusted so that FVIIa elutes with a retention time of approximately 26minutes. Flow rate: 1.0 mL/min. Detection: 214 nm. Load: 25 μg FVIIa.

Example 2

In order to investigate the effect of arginine on the stability ofrFVIIa the following solutions were prepared:

Formulation 5:

-   1.0 mg/mL rFVIIa-   25 mM HEPES-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.5    Formulation 6:-   1.0 mg/mL rFVIIa-   25 mM HEPES-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   200 mM Arginine-   pH=7.5    Formulation 7-   1.0 mg/mL rFVIIa-   20 mM Histidine-   20 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.0    Formulation 8-   1.0 mg/mL rFVIIa-   50 mM Histidine-   50 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   200 mM Arginine-   pH=7.0    Formulation 9-   1.0 mg/mL rFVIIa-   50 mM Histidine-   50 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   400 mM Arginine-   pH=7.0

The formulations were prepared by adding 25 mM HEPES (solutions 5 and 6)and arginine (solutions 6, 8 and 9) to a 1.0 mg/mL bulk solution ofrFVIIa already containing glycylglycine, sodium chloride and calciumchloride in the above mentioned concentrations. pH was finally adjustedwith 1 M sodium hydroxide and 1 M hydrochloric acid.

The formulations were stored at a temperature of 5° C. and 30° C., andthe analyses for formation of heavy chain degradation products wereperformed as in Example 1 at time points shown in the Table 2.

TABLE 2 Formation of heavy chain degradation products (hcd) in arginineformulations % hcd % hcd % hcd % hcd % hcd ½ months 1 months 2 months 3months Formulation 0 months 30° C. 5° C. 30° C. 5° C. 30° C. 5° C. 30°C. 5° C. 5 17.3 28.7 32.0 35.5 42.7 42.1 57.0 44.4 64.3 6 13.6 19.5 21.225.5 29.8 30.9 41.9 34.0 49.0 7 9.4 — n.a. — 31.7 — 44.6 — 52.5 8 11.7*— 17.3 — 23.0 — 31.1 — 37.7 9 11.5* — 14.0 — 17.4 — 21.5 — 25.4 analysedat t = 3 days, a slight increase is expected from day 0 to day 3 n.a.:not analysed

Example 3

Formulation of the following liquid, aqueous pharmaceutical compositionsis envisaged:

A) rhFVIIa 1 mg/mL (approx. 50,000 IU/mL) PIPES 15.12 mg/mL (50 mM)Benzamidine 50 mM Poloxamer 188 0.5 mg/mL Sodium chloride 2.92 mg/mL (50mM) Calcium chloride 2 H₂O 1.47 mg/mL (10 mM) Methionine 0.5 mg/mL 1MNaOH/1M HCl added to pH 6.5 B) rhFVIIa 1 mg/mL (approx. 50,000 IU/mL)PIPES 15.12 mg/mL (50 mM) p-Aminobenzamidine 10 mM Poloxamer 188 0.5mg/mL Sodium chloride 2.92 mg/mL (50 mM) Calcium chloride 2 H₂O 1.47mg/mL (10 mM) Methionine 0.5 mg/mL 1M NaOH/1M HCl added to pH 6.5 C)rhFVIIa 1 mg/mL (approx. 50,000 IU/mL) PIPES 15.12 mg/mL (50 mM)Arginine 50 mM Poloxamer 188 0.5 mg/mL Sodium chloride 2.92 mg/mL (50mM) Calcium chloride 2 H₂O 1.47 mg/mL (10 mM) 1M NaOH/1M HCl added to pH6.5 D) rhFVIIa 1 mg/mL (approx. 50,000 IU/mL) PIPES 15.12 mg/mL (50 mM)Arginine 100 mM Poloxamer 188 0.5 mg/mL Sodium chloride 2.92 mg/mL (50mM) Calcium chloride 2 H₂O 1.47 mg/mL (10 mM) 1M NaOH/1M HCl added to pH6.5

Pharmaceutical compositions A-D can subsequently be transferred tosterile vials or cartridges flushed with nitrogen or argon and can thenbe packed in air-tight aluminium-laminated plastic bags.

Example 4

In order to investigate the effect of benzamidine on the stability ofrFVIIa the following formulations were prepared:

Formulation 1

-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=6.5    Formulation 2-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 mM Benzamidine-   pH=6.5    Formulation 3-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.5    Formulation 4-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 mM Benzamidine-   pH=7.5

The formulations were prepared by adding 10 mM histidine and 50 mMbenzamidine (only for formulations 2 and 4) to a 1.0 mg/mL bulk solutionof rFVIIa already containing glycylglycine, sodium chloride and calciumchloride in the above-mentioned concentrations. pH was finally adjustedto 6.5 and 7.5, respectively, with 1 M sodium hydroxide and 1 Mhydrochloric acid.

The formulations were stored at a temperature of 5° C. and the analysesfor clot activity were performed at time points shown in Table 3:

TABLE 3 Clot activity (IU/mL) Storage time at 5° C. (months) Formulation0 3 7 10 1 46,200 31,000 <1,000 Not analysed 2 43,300 43,800 44,40043,200 3 42,800 14,900 9,700 Not analysed 4 44,700 40,000 40,600 39,300

Example 5

In order to investigate the effect of different stabilizers on thestability of rFVIIa the following formulations were prepared:

Formulation 1

-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=6.5    Formulation 2-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   5 mM p-amino-Benzamidine-   pH=6.5    Formulation 3-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   0.5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=6.5    Formulation 4-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.5    Formulation 5-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   5 mM p-amino-Benzamidine

pH=7.5

Formulation 6

-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   0.5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=7.5

The formulations were prepared by adding 10 mM histidine, 5 mMp-amino-Benzamidine (for formulations 2 and 5) and 0.5 mMS-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide(for formulations 3 and 6) to a 1.0 mg/mL bulk solution of rFVIIaalready containing glycylglycine, sodium chloride and calcium chloridein the above-mentioned concentrations. pH was finally adjusted to 6.5and 7.5, respectively, with 1 M sodium hydroxide and 1 M hydrochloricacid.

The formulations were stored at a temperature of 5° C. and the analysesfor clot activity (Table 4) and Heavy chain degradation (Table 5) wereperformed at the time points shown in the tables:

TABLE 4 Clot activity (IU/mL) Storage time at 5° C. (months) Formulation0 3 6 9 1 42,400 29,300 23,500 20,600 2 40,700 46,700 39,500 41,200 343,400 42,400 39,700 44,200 4 38.900 16,200 9,800 Not analysed 5 44,90042,500 37,800 35,900 6 39,800 41,200 45,700 43,100

TABLE 5 Content of Heavy chain degradation (%) Storage time at 5° C.(months) Formulation 0 1 3 6 1 12.9 23.6 38.0 51.1 2 12.1 12.5 13.4 15.23 12.1 11.5 10.9 12.0 4 16.9 54.0 71.5 76.7 5 12.7 16.7 22.0 29.1 6 11.911.3 11.3 11.7

Example 6

In order to investigate the effect of benzamidine andN-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide, HCl onthe stability of rFVIIa the following formulations were prepared:

Formulation 1

-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=6.5    Formulation 2-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 mM Benzamidine-   pH=6.5    Formulation 3-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 μM    N-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide HCl-   pH=6.5    Formulation 4-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   500 μM    N-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide HCl-   pH=6.5    Formulation 5-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.5    Formulation 6-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 mM Benzamidine-   pH=7.5    Formulation 7-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   50 μM    N-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide HCl-   pH=7.5    Formulation 8-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   500 μM    N-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide HCl-   pH=7.5

The formulations were prepared by adding 10 mM histidine, 50 mMbenzamidine (for formulations 2 and 6) and 50/500 μMN-(3-Bromobenzyl)-2-[3-(4-carbamimidoylphenyl)-ureido]-acetamide HCl(for formulation 3, 4 and 7,8 to a 1.0 mg/mL bulk solution of rFVIIaalready containing glycylglycine, sodium chloride and calcium chloridein the above-mentioned concentrations. pH was finally adjusted to 6.5and 7.5, respectively, with 1 M sodium hydroxide and 1 M hydrochloricacid.

The formulations were stored at a temperature of 5° C. and 30° C. andthe analyses for Clot activity were performed at the time points shownin table 6:

TABLE 6 Clot activity (IU/mL) Storage time at 5° C. (months) Formulation0 3 7 10 12 1 46,200 31,000 <1,000 Not analysed Not analysed 2 43,30043,800 44,400 43,200 45,000 3 44,300 36,700 33,700 * 31,200 4 46,00037,800 35,600 * 36,000 5 42,800 14,900 9,700 Not analysed Not analysed 644,700 40,000 40,600 39,300 40,500 7 44,100 35,800 32,700 29,200 * 843,300 40,800 39,400 * 36,600 Storage time at 30° C. (months)Formulation 0 1 2 1 46,200 26,200 16,900 2 43,300 38,100 31,000 3 44,30025,800 17,500 4 46,000 22,000 14,900 5 42,800 17,200 10,500 6 44,70028,800 19,000 7 44,100 17,900 13,400 8 43,300 23,900 17,100 *:unreliable result, not reported

Example 7

In order to investigate the effect of different stabilisers on thestability of rFVIIa the following formulations were prepared:

Formulation 1

-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=6.5    Formulation 2-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   5 mM p-amino-Benzamidine-   pH=6.5    Formulation 3-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   0,05 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=6.5    Formulation 4-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   0,5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=6.5    Formulation 5-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   pH=7.5    Formulation 6-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   5 mM p-amino-Benzamidine-   pH=7.5    Formulation 7-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   0,05 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=7.5    Formulation 8-   1.0 mg/mL rFVIIa-   10 mM Histidine-   10 mM Glycylglycine-   50 mM Sodium chloride-   10 mM Calcium chloride-   0,5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=7.5

The formulations were prepared by adding 10 mM histidine, 5 mMp-amino-Benzamidine (for formulations 2 and 6) and 0,05/0,5 mMS-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide(for formulations 3, 7 and 4, 8) to a 1.0 mg/mL bulk solution of rFVIIaalready containing glycylglycine, sodium chloride and calcium chloridein the above-mentioned concentrations. pH was finally adjusted to 6.5and 7.5, respectively, with 1 M sodium hydroxide and 1 M hydrochloricacid.

The formulations were stored at a temperature of 5° C. and the analysesfor Clot activity (table 7) and Heavy chain degradation (table 8) wereperformed at the time points shown in the tables:

TABLE 7 Clot activity (IU/mL) Storage time at 5° C. (months) Formulation0 3 6 9 1 42,400 29,300 23,500 20,600 2 40,700 46,700 39,500 41,200 339.600 40,800 40,200 40,100 4 43,400 42,400 39,700 44,200 5 38,90016,200 9,800 Not analysed 6 44,900 42,500 37,800 35,900 7 39,100 39,10040,200 38,900 8 39,800 41,200 45,700 43,100 Storage time at 30° C.(months) Formulation 0 2 3 1 42,400 15,000 8,700 2 40,700 21,600 15,4003 39.600 19,100 12,000 4 43,400 29,700 25,400 5 38.900 11,000 6,300 644,900 18,300 11,100 7 39,100 21,600 15,100 8 39,800 34,800 27,300

TABLE 8 Content of Heavy chain degradation (%) Storage time at 5° C.(months) Formulation 0 1 3 6 1 12.9 23.6 38.0 51.1 2 12.1 12.5 13.4 15.23 12.2 13.0 15.0 18.5 4 12.1 11.5 10.9 12.0 5 16.9 54.0 71.5 76.7 6 12.716.7 22.0 29.1 7 12.4 13.3 13.8 17.2 8 11.9 11.3 11.3 11.7 Storage timeat 30° C. (months) Formulation 0 1 2 3 1 12.9 23.3 26.3 29.0 2 12.1 20.223.2 26.7 3 12.2 20.3 23.8 25.9 4 12.1 14.8 16.4 18.6 5 16.9 41.1 47.749.2 6 12.7 34.8 41.5 46.3 7 12.4 32.3 39.1 43.4 8 11.9 14.5 16.3 18.1

Example 8

In order to investigate the effect ofS-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamideon the stability of rFVIIa the following formulations were prepared:

Formulation 1

-   1.0 mg/mL rFVIIa-   50 mM Sodium chloride-   10 mM Calcium chloride-   10 mM Glycylglycine-   20 mM Histidine-   0.5 mg/mL Methionine-   5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=6.0    Formulation 2-   1.0 mg/mL rFVIIa-   50 mM Sodium chloride-   10 mM Calcium chloride-   10 mM Glycylglycine-   20 mM Histidine-   0.5 mg/mL Methionine-   5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=6.5    Formulation 3-   1.0 mg/mL rFVIIa-   50 mM Sodium chloride-   10 mM Calcium chloride-   10 mM Glycylglycine-   20 mM Histidine-   0.5 mg/mL Methionine-   5 mM    S-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamide-   pH=7.5

The formulations were prepared by adding 20 mM histidine, 5 mMS-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamideand 0.5 mg/mL Methionine to a solution of rFVIIa already containingglycylglycine, sodium chloride and calcium chloride in theabove-mentioned concentrations. pH was finally adjusted to 6.0, 6.5 and7.5, respectively, with 1 M sodium hydroxide/ hydrochloric acid.

The formulations were stored at a temperature of 25° C. and the analysesfor Clot activity (table 9) and Heavy chain degradation (table 10) wereperformed at the time points shown in the tables:

TABLE 9 Clot activity (IU/mL) Storage time at Storage time at 25° C. 5°C. (months) (months) Formulation 0 3 6 3 1 38,400 45,500 41,700 38,500 239,800 41,200 37,800 42,400 3 42,400 43,000 39,500 39,900

The reference formulations withoutS-2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[1-(3-methoxyphenyl)-ethyl]-acetamideshows the following Clot activity.

TABLE 10 Clot activity (IU/mL) for reference solutions Storage time atStorage time at 25° C. Reference 5° C. (months) (months) Formulation 0 13 3 1 38,400* 44,400 21,300 4,400 2 39,800* 40,100 15,400 2,500 342,400* 26,400 7,700 <1,000 *The time zero results for the referencesolutions are not available, for this reason the corresponding valuesfor the solutions containing the inhibitor substance has been listed.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

1. A liquid, aqueous, and pharmaceutically acceptable compositioncomprising (i) at least 0.01 mg/mL of a Factor VII polypeptide; (ii) abuffering agent suitable for keeping pH of the composition in the rangeof about 4 to about 9; and (iii) a stabilizing composition comprising abenzamidine compound comprising a motif comprising—substituted benzenering—C(═N—Z¹—R¹)—NH—Z²—R², wherein substituted benzene ring denotes abenzene ring comprising one substitution, and Z¹ and Z² independentlyare selected from the group consisting of —O—, —S—, —NR^(H)— and asingle bond, where R^(H) is selected from the group consisting ofhydrogen, C₁₋₄-alkyl, aryl and arylmethyl, and R¹ and R² independentlyare selected from the group consisting of hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted aryl, optionally substituted heterocyclyl, —C═N—Z¹—R¹ formspart of a heterocyclic ring, —C—NH—Z²—R² forms part of a heterocyclicring, or —C(═N—Z¹—R¹)—NH—Z²—R² forms a hetercyclic ring wherein—Z¹—R¹—R²—Z²— is a biradical.
 2. The composition of claim 1, wherein themolecular weight of the benzamidine compound is 1000 Da or less.
 3. Thecomposition of claim 2, wherein the benzamidine is p-amino-benzamidine.4. The composition of claim 3, wherein the concentration ofp-amino-benzamidine is at least 1 μM.
 5. The composition of claim 2,wherein the benzamidine is m-amino-benzamidine.
 6. The composition ofclaim 2, wherein the benzamidine is p-amino-benzamidine.
 7. Thecomposition of claim 1, wherein the concentration of the benzan compoundis at least 1 μM.
 8. The composition of claim 7, wherein theconcentration of the benzamidine compound is at least 1 mM.
 9. Thecomposition according to claim 1, wherein the benzamidine compound is2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[(S)-1-(3-methoxyphenyl)-ethyl]-acetamide.10. The composition of claim 9, wherein the concentration of2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[(S)-1-(3-methoxyphenyl)-ethyl]-acetamideis at least 1 μM.
 11. The composition of claim 1, wherein the Factor VIIpolypeptide is human Factor VIIa.
 12. The composition of claim 1,wherein the Factor VII polypeptide is a Factor VII sequence variant. 13.The composition of claim 1, wherein the Factor VII polypeptide ispresent in a concentration of 0.01-20 mg/mt.
 14. The composition ofclaim 1, wherein the buffering agent (ii) comprises at least onecomponent selected from the group consisting of acids and salts of MES,PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine,glycylglycine, glycinamide, phosphoric acid, acetic acid, lactic acid,glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, andsuccinic acid.
 15. The composition of claim 14, wherein theconcentration of the buffering agent (ii) is about 1-100 mM.
 16. Thecomposition of claim 1, wherein the composition further comprises anon-ionic surfactant (iv).
 17. The composition of claim 16, wherein thenon-ionic surfactant (iv) is at least one selected from the groupconsisting of polysorbates, poloxamers, polyoxyethylene alkyl ethers,ethylene/polypropylene block co-polymers and polyethyleneglycol (PEG).18. The composition of claim 17, wherein the concentration of thenon-ionic surfactant (iv) is 0.005-2% by weight.
 19. The composition ofclaim 16, wherein the composition further comprises a tonicity modifyingagent (v).
 20. The composition of claim 19, wherein the tonicitymodifying agent (v) is at least one selected from the group consistingof neutral salts, amino acids, peptides of 2-5 amino acid residues,monosaccharides, disaccharides, polysaccharides, and sugar alcohols. 21.The composition of claim 20, wherein the tonicity modifying agent (v) isa neutral salt selected from the group consisting of sodium salts,potassium salts, calcium salts, and magnesium salts.
 22. The compositionof claim 21, wherein the composition further comprises an antioxidant(vi).
 23. The composition of claim 22, wherein the antioxidant (vi) isselected from L-methionine, D-methionine, methionine analogues,methionine-containing peptides, ascorbic acid, cysteine, homocysteine,gluthatione, cystine, and cystathionine.
 24. The composition of claim23, wherein the antioxidant (vi) is present in a concentration of0.1-5.0 mg/mL.
 25. The composition of claim 22, wherein the compositionfurther comprises a preservative (vii).
 26. The composition of claim 25,wherein the preservative (vii) is selected from the group consisting ofphenol, benzyl alcohol, orto-cresol, meta-cresol, para-cresol, methylparaben, propyl paraben, benzalkonium chloride, and benzaethoniumchloride.
 27. The composition of claim 20, wherein the tonicitymodifying agent (v) is sodium chloride in combination with at least oneselected from the group consisting of calcium chloride, calcium acetate,magnesium chloride and magnesium acetate.
 28. The composition of claim27, wherein the tonicity modifying, agent (v) is present in aconcentration of at least 1 mM.
 29. The composition of claim 19, whereinthe tonicity modifying agent (v) is an ionic strength modifying agent.30. The composition of claim 29, wherein the composition has an ionicstrength of at least 50 mM.
 31. The composition of claim 1, wherein thecomposition further comprises a tonicity modifying agent (v).
 32. Thecomposition according to claim 1, wherein the composition has anosmolality of 300±50 milliosmol/kg.
 33. The composition of claim 1,wherein the composition further comprises an antioxidant (vi).
 34. Thecomposition of claim 1, wherein the composition further comprises apreservative (vii).
 35. The composition of claim 1, wherein thecomposition comprises 0.1-20 mg/mL of a Factor VII polypeptide; thestabilizing agent comprises an effective amount of p-amino-herizamidine,2-[3-(4-Carbamimidoylphenyl)-ureido]-N-[(S)-1-(3-methoxyphenyl)-ethyl]-acetamide,or a mixture thereof, in a concentration of at least 5 μM; and thecomposition further comprises a non-ionic surfactant (iv) and a tonicitymodifying agent (v) in a concentration of at least 5 μM.
 36. Thecomposition of claim 1, wherein the composition is adapted forparenteral administration.
 37. The composition of claim 1, wherein thecomposition is adapted for subcutaneous, intramuscular, or intravenousinjection.
 38. An air-tight container containing the composition ofclaim 1, and optionally an inert gas.
 39. The container of claim 38,wherein the composition does not comprise an antioxidant.